A Complex Connection Between the Diversity of Human Gastric Mucin O-Glycans, Helicobacter pylori Binding, Helicobacter Infection and Fucosylation.

Helicobacter pylori colonizes the stomach of half of the human population. Most H. pylori are located in the mucus layer, which is mainly comprised by glycosylated mucins. Using mass spectrometry, we identified 631 glycans (whereof 145 were fully characterized and the remainder assigned as compositions) on mucins isolated from 14 Helicobacter spp.-infected and 14 Helicobacter spp.-noninfected stomachs. Only six identified glycans were common to all individuals, from a total of 60 to 189 glycans in each individual. An increased number of unique glycan structures together with an increased intraindividual diversity and larger interindividual variation were identified among O-glycans from Helicobacter spp.-infected stomachs compared with noninfected stomachs. H. pylori strain J99, which carries the blood group antigen-binding adhesin (BabA), the sialic acid-binding adhesin (SabA), and the LacdiNAc-binding adhesin, bound both to Lewis b (Leb)-positive and Leb-negative mucins. Among Leb-positive mucins, H. pylori J99 binding was higher to mucins from Helicobacter spp.-infected individuals than noninfected individuals. Statistical correlation analysis, binding experiments with J99 wt, and J99ΔbabAΔsabA and inhibition experiments using synthetic glycoconjugates demonstrated that the differences in H. pylori-binding ability among these four groups were governed by BabA-dependent binding to fucosylated structures. LacdiNAc levels were lower in mucins that bound to J99 lacking BabA and SabA than in mucins that did not, suggesting that LacdiNAc did not significantly contribute to the binding. We identified 24 O-glycans from Leb-negative mucins that correlated well with H. pylori binding whereof 23 contained α1,2-linked fucosylation. The large and diverse gastric glycan library identified, including structures that correlated with H. pylori binding, could be used to select glycodeterminants to experimentally investigate further for their importance in host-pathogen interactions and as candidates to develop glycan-based therapies.

Atlantic Salmon Mucins Inhibit LuxS-Dependent A. Salmonicida AI-2 Quorum Sensing in an N-Acetylneuraminic Acid-Dependent Manner.

One of the most important bacterial diseases in salmonid aquaculture is furunculosis, caused by Aeromonas salmonicida. Bacterial communication through secreted autoinducer signals, quorum sensing, takes part in the regulation of gene expression in bacteria, influencing growth and virulence. The skin and mucosal surfaces, covered by a mucus layer, are the first point of contact between fish and bacteria. Mucins are highly glycosylated and are the main components of mucus. Here, we validate the Vibrio harveyi BB170 bioreporter assay for quantifying A. salmonicida quorum sensing and study the effects of Atlantic salmon mucins as well as mono- and disaccharides on the AI-2 levels of A. salmonicida. Atlantic salmon mucins from skin, pyloric ceca, proximal and distal intestine reduced A. salmonicida AI-2 levels. Among the saccharides abundant on mucins, fucose, N-acetylneuraminic acid and GlcNAcß1-3Gal inhibited AI-2 A. salmonicida secretion. Removal of N-acetylneuraminic acid, which is the most abundant terminal residue on mucin glycans on Atlantic salmon mucins, attenuated the inhibitory effects on AI-2 levels of A. salmonicida. Deletion of A. salmonicida luxS abolished AI-2 production. In conclusion, Atlantic salmon mucins regulate A. salmonicida quorum sensing in a luxS and N-acetylneuraminic acid-dependent manner.

Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins.

Sulfomucins are in some body locations and species a normal occurrence, whereas in other situations, are a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH 1.0. However, since the stain detects the charge, a high density of other charged molecules, such as sialic acids, has potential to impede specificity. Here, we compared the outcome from four staining protocols with the level of sulfation determined by liquid chromatography-tandem mass spectrometric analysis on samples from various tissues with variable sulfation and sialylation levels. We found that a protocol we designed, including rinsing with MetOH and 0.5 M NaCl buffer at pH 1.0, eliminates the false positive staining of tissues outperforming commonly recommended solutions. In tissues with low-to-moderately sulfated mucins (e.g. human stomach and salmonid epithelia), this method enables accurate relative quantification (e.g. sulfate scoring comparisons between healthy and diseased tissues), whereas the range of the method is not suitable for comparisons between tissues with high sulfomucin content (e.g. pig stomach and colon).

Mucin Binding to Moraxella catarrhalis during Airway Inflammation Is Dependent on Sialic Acid.

Chronic obstructive pulmonary disease (COPD) is associated with colonization by bacterial pathogens and repeated airway infections, leading to exacerbations and impaired lung function. The highly glycosylated mucins in the mucus lining the airways are an important part of the host defense against pathogens. However, mucus accumulation can contribute to COPD pathology. Here, we examined whether inflammation is associated with glycosylation changes that affect interactions between airway mucins and pathogens. We isolated mucins from lower airway samples (n = 4-9) from long-term smokers with and without COPD and from never-smokers. The most abundant terminal glycan moiety was N-acetylneuraminic acid (Neu5Ac) among smokers with and without COPD and N-acetyl-hexoseamine among never-smokers. Moraxella catarrhalis bound to MUC5 mucins from smokers with and without COPD. M. catarrhalis binding correlated with inflammatory parameters and Neu5Ac content. M. catarrhalis binding was abolished by enzymatic removal of Neu5Ac. Furthermore, M. catarrhalis bound to α2,6 sialyl-lactose, suggesting that α2,6 sialic acid contributes to M. catarrhalis binding to mucins. Furthermore, we detected more M. catarrhalis binding to mucins from patients with pneumonia than to those from control subjects (n = 8-13), and this binding correlated with C-reactive protein and Neu5Ac levels. These results suggest a key role of inflammation-induced Neu5Ac in the adhesion of M. catarrhalis to airway mucins. The inflammation-induced ability of MUC5 mucins to bind M. catarrhalis is likely a host defense mechanism in the healthy lung, although it cannot be excluded that impaired mucociliary clearance limits the effectiveness of this defense in patients with COPD.

Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar.

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O-glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14-30 kg/m3) and high densities (HD: 50-80 kg/m3) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (Papp mannitol; p < 0.01). Liquid chromatography-mass spectrometry revealed 81 O-glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O-glycan structures. Overall, four of the O-glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators.

Gill Mucus and Gill Mucin O-glycosylation in Healthy and Amebic Gill Disease-Affected Atlantic Salmon.

Amoebic gill disease (AGD) causes poor performance and death in salmonids. Mucins are mainly comprised by carbohydrates and are main components of the mucus covering the gill. Since glycans regulate pathogen binding and growth, glycosylation changes may affect susceptibility to primary and secondary infections. We investigated gill mucin O-glycosylation from Atlantic salmon with and without AGD using liquid chromatography-mass spectrometry. Gill mucin glycans were larger and more complex, diverse and fucosylated than skin mucins. Confocal microscopy revealed that fucosylated mucus coated sialylated mucus strands in ex vivo gill mucus. Terminal HexNAcs were more abundant among O-glycans from AGD-affected Atlantic salmon, whereas core 1 structures and structures with acidic moieties such as N-acetylneuraminic acid (NeuAc) and sulfate groups were less abundant compared to non-infected fish. The fucosylated and NeuAc-containing O-glycans were inversely proportional, with infected fish on the lower scale of NeuAc abundance and high on fucosylated structures. The fucosylated epitopes were of three types: Fuc-HexNAc-R, Gal-[Fuc-]HexNAc-R and HexNAc-[Fuc-]HexNAc-R. These blood group-like structures could be an avenue to diversify the glycan repertoire to limit infection in the exposed gills. Furthermore, care must be taken when using skin mucus as proxy for gill mucus, as gill mucins are distinctly different from skin mucins.

Recombinant mucin-type proteins carrying LacdiNAc on different O-glycan core chains fail to support H. pylori binding.

The ß4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a ß1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment of Helicobacter pylori to the gastric mucosa via binding to the LDN determinant. The O-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried by O-glycan core 2, core 3 and extended core 1 precursors using transient transfection of CHO-K1 cells and a mucin-type immunoglobulin fusion protein as reporter protein. Binding of the LabA-positive H. pylori J99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on different O-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of 125I-LDN-BSA to various clinical H. pylori isolates was assessed in solution. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blotting confirmed the requirement of a terminal GlcNAc for B4GALNT3 activity. B4GALNT3 added a ß1,4-linked GalNAc to GlcNAc irrespective of whether the latter was carried by a core 2, core 3 or extended core 1 chain. No LDN-mediated adhesion of H. pylori strains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1 O-glycans were detected in a microtiter well-based adhesion assay and no binding of a 125I-labelled LDN-BSA neoglycoconjugate to clinical H. pylori isolates was identified.

Mucin modified SPR interfaces for studying the effect of flow on pathogen binding to Atlantic salmon mucins.

Knowledge on host-pathogen interactions contributes to the development of approaches to alleviate infectious disease. In this work, we developed a surface plasmon resonance (SPR) based method for investigating bacteria/mucins interactions. Furthermore, we investigated adhesion of three pathogens, Aeromonas salmonicida, Aeromonas hydrophila and Vibrio harveyi, to Atlantic salmon mucins isolated from different epithelial sites, using SPR and microtiter-based binding assays. We demonstrated that performing bacterial binding assays to mucins using SPR is feasible and has advantages over microtiter-based binding assays, especially under flow conditions. The fluid flow in the SPR is linear and continuous and SPR enables real-time reading of mucin-bacterial bonds, which provides an in vivo-like setup for analysis of bacterial binding to mucins. The variation between technical replicates was smaller using SPR detection compared to the adenosine 5'-triphosphate (ATP) bioluminescence assay in microtiter plates. Furthermore, we demonstrated that the effect of flow on pathogen-mucin interaction is significant and that bacterial adhesion differ non-linearly with flow rates and depend on the epithelial source of the mucin.

The Farmed Atlantic Salmon (Salmo salar) Skin-Mucus Proteome and Its Nutrient Potential for the Resident Bacterial Community.

Norway is the largest producer and exporter of farmed Atlantic salmon (Salmo salar) worldwide. Skin disorders correlated with bacterial infections represent an important challenge for fish farmers due to the economic losses caused. Little is known about this topic, thus studying the skin-mucus of Salmo salar and its bacterial community depict a step forward in understanding fish welfare in aquaculture. In this study, we used label free quantitative mass spectrometry to investigate the skin-mucus proteins associated with both Atlantic salmon and bacteria. In particular, the microbial temporal proteome dynamics during nine days of mucus incubation with sterilized seawater was investigated, in order to evaluate their capacity to utilize mucus components for growth in this environment. At the start of the incubation period, the largest proportion of proteins (~99%) belonged to the salmon and many of these proteins were assigned to protecting functions, confirming the defensive role of mucus. On the contrary, after nine days of incubation, most of the proteins detected were assigned to bacteria, mainly to the genera Vibrio and Pseudoalteromonas. Most of the predicted secreted proteins were affiliated with transport and metabolic processes. In particular, a large abundance and variety of bacterial proteases were observed, highlighting the capacity of bacteria to degrade the skin-mucus proteins of Atlantic salmon.

Fish pathogen binding to mucins from Atlantic salmon and Arctic char differs in avidity and specificity and is modulated by fluid velocity.

Disease outbreaks are limiting factors for an ethical and economically sustainable aquaculture industry. The first point of contact between a pathogen and a host occurs in the mucus, which covers the epithelial surfaces of the skin, gills and gastrointestinal tract. Increased knowledge on host-pathogen interactions at these primary barriers may contribute to development of disease prevention strategies. The mucus layer is built of highly glycosylated mucins, and mucin glycosylation differs between these epithelial sites. We have previously shown that A. salmonicida binds to Atlantic salmon mucins. Here we demonstrate binding of four additional bacteria, A. hydrophila, V. harveyi, M. viscosa and Y. ruckeri, to mucins from Atlantic salmon and Arctic char. No specific binding could be observed for V. salmonicida to any of the mucin groups. Mucin binding avidity was highest for A. hydrophila and A. salmonicida, followed by V. harveyi, M. viscosa and Y. ruckeri in decreasing order. Four of the pathogens showed highest binding to either gills or intestinal mucins, whereas none of the pathogens had preference for binding to skin mucins. Fluid velocity enhanced binding of intestinal mucins to A. hydrophila and A. salmonicida at 1.5 and 2 cm/s, whereas a velocity of 2 cm/s for skin mucins increased binding of A. salmonicida and decreased binding of A. hydrophila. Binding avidity, specificity and the effect of fluid velocity on binding thus differ between salmonid pathogens and with mucin origin. The results are in line with a model where the short skin mucin glycans contribute to contact with pathogens whereas pathogen binding to mucins with complex glycans aid the removal of pathogens from internal epithelial surfaces.

Exploring the Arctic Charr Intestinal Glycome: Evidence of Increased N-Glycolylneuraminic Acid Levels and Changed Host-Pathogen Interactions in Response to Inflammation.

Disease outbreaks are a limiting factor for the sustainable development of the aquaculture industry. The intestinal tract is covered by a mucus layer mainly comprised by highly glycosylated proteins called mucins. Mucins regulate pathogen adhesion, growth, and virulence, and the glycans are vital for these functions. We analyzed intestinal mucin O-glycans on mucins from control and full-fat extruded soy-bean-fed (known to cause enteritis) Arctic charr using liquid chromatography-tandem mass spectrometry. In total, 56 glycans were identified on Arctic charr intestinal mucins, with a high prevalence of core-5-type and sialylated O-glycans. Disialic-acid-epitope-containing structures including NeuAcα2,8NeuAc, NeuAc(Gc)α2,8NeuGc(Ac), and NeuGcα2,8NeuGc were the hallmark of Arctic charr intestinal mucin glycosylation. Arctic charr fed with soy bean meal diet had lower (i) number of structures detected, (ii) interindividual variation, and (iii) N-glycolylneuraminic-acid-containing glycans compared with control Arctic charr. Furthermore, Aeromonas salmonicida grew less in response to mucins from inflamed Arctic charr than from the control group. The Arctic charr glycan repertoire differed from that of Atlantic salmon. In conclusion, the loss of N-glycolylneuraminic acid may be a biomarker for inflammation in Arctic char, and inflammation-induced glycosylation changes affect host-pathogen interactions.

Effects of Size and Geographical Origin on Atlantic salmon, Salmo salar, Mucin O-Glycan Repertoire.

Diseases cause ethical concerns and economic losses in the Salmonid industry. The mucus layer comprised of highly O-glycosylated mucins is the first contact between pathogens and fish. Mucin glycans govern pathogen adhesion, growth and virulence. The Atlantic salmon O-glycome from a single location has been characterized and the interindividual variation was low. Because interindividual variation is considered a population-based defense, hindering the entire population from being wiped out by a single infection, low interindividual variation among Atlantic salmon may be a concern. Here, we analyzed the O-glycome of 25 Atlantic salmon from six cohorts grown under various conditions from Sweden, Norway and Australia (Tasmania) using mass spectrometry. This expanded the known Atlantic salmon O-glycome by 60% to 169 identified structures. The mucin O-glycosylation was relatively stable over time within a geographical region, but the size of the fish affected skin mucin glycosylation. The skin mucin glycan repertoires from Swedish and Norwegian Atlantic salmon populations were closely related compared with Tasmanian ones, regardless of size and salinity, with differences in glycan size and composition. The internal mucin glycan repertoire also clustered based on geographical origin and into pyloric cecal and distal intestinal groups, regardless of cohort and fish size. Fucosylated structures were more abundant in Tasmanian pyloric caeca and distal intestine mucins compared with Swedish ones. Overall, Tasmanian Atlantic salmon mucins have more O-glycan structures in skin but less in the gastrointestinal tract compared with Swedish fish. Low interindividual variation was confirmed within each cohort. The results can serve as a library for identifying structures of importance for host-pathogen interactions, understanding population differences of salmon mucin glycosylation in resistance to diseases and during breeding and selection of strains. The results could make it possible to predict potential vulnerabilities to diseases and suggest that inter-region breeding may increase the glycan diversity.

Mucus-Pathogen Interactions in the Gastrointestinal Tract of Farmed Animals.

Gastrointestinal infections cause significant challenges and economic losses in animal husbandry. As pathogens becoming resistant to antibiotics are a growing concern worldwide, alternative strategies to treat infections in farmed animals are necessary in order to decrease the risk to human health and increase animal health and productivity. Mucosal surfaces are the most common route used by pathogens to enter the body. The mucosal surface that lines the gastrointestinal tract is covered by a continuously secreted mucus layer that protects the epithelial surface. The mucus layer is the first barrier the pathogen must overcome for successful colonization, and is mainly composed of densely glycosylated proteins called mucins. The vast array of carbohydrate structures present on the mucins provide an important setting for host-pathogen interactions. This review summarizes the current knowledge on gastrointestinal mucins and their role during infections in farmed animals. We examine the interactions between mucins and animal pathogens, with a focus on how pathogenic bacteria can modify the mucin environment in the gut, and how this in turn affects pathogen adhesion and growth. Finally, we discuss analytical challenges and complexities of the mucus-based defense, as well as its potential to control infections in farmed animals.

Helicobacter suis binding to carbohydrates on human and porcine gastric mucins and glycolipids occurs via two modes.

Helicobacter suis colonizes the stomach of most pigs and is the most prevalent non-Helicobacter pylori Helicobacter species found in the human stomach. In the human host, H. suis contributes to the development of chronic gastritis, peptic ulcer disease and MALT lymphoma, whereas in pigs it is associated with gastritis, decreased growth and ulcers. Here, we demonstrate that the level of H. pylori and H. suis binding to human and pig gastric mucins varies between individuals with species dependent specificity. The binding optimum of H. pylori is at neutral pH whereas that of H. suis has an acidic pH optimum, and the mucins that H. pylori bind to are different than those that H. suis bind to. Mass spectrometric analysis of mucin O-glycans from the porcine mucin showed that individual variation in binding is reflected by a difference in glycosylation; of 109 oligosaccharide structures identified, only 14 were present in all examined samples. H. suis binding to mucins correlated with glycans containing sulfate, sialic acid and terminal galactose. Among the glycolipids present in pig stomach, binding to lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer) was identified, and adhesion to Galß3GlcNAcß3Galß4Glc at both acidic and neutral pH was confirmed using other glycoconjugates. Together with that H. suis bound to DNA (used as a proxy for acidic charge), we conclude that H. suis has two binding modes: one to glycans terminating with Galß3GlcNAc, and one to negatively charged structures. Identification of the glycan structures H. suis interacts with can contribute to development of therapeutic strategies alternative to antibiotics.

Aeromonas salmonicida Growth in Response to Atlantic Salmon Mucins Differs between Epithelial Sites, Is Governed by Sialylated and N-Acetylhexosamine-Containing O-Glycans, and Is Affected by Ca2.

Aeromonas salmonicida causes furunculosis in salmonids and is a threat to Atlantic salmon aquaculture. The epithelial surfaces that the pathogen colonizes are covered by a mucus layer predominantly comprised of secreted mucins. By using mass spectrometry to identify mucin glycan structures with and without enzymatic removal of glycan residues, coupled to measurements of bacterial growth, we show here that the complex Atlantic salmon intestinal mucin glycans enhance A. salmonicida growth, whereas the more simple skin mucin glycans do not. Of the glycan residues present terminally on the salmon mucins, only N-acetylglucosamine (GlcNAc) enhances growth. Sialic acids, which have an abundance of 75% among terminal glycans from skin and of <50% among intestinal glycans, cannot be removed or used by A. salmonicida for growth-enhancing purposes, and they shield internal GlcNAc from utilization. A Ca2+ concentration above 0.1 mM is needed for A. salmonicida to be able to utilize mucins for growth-promoting purposes, and 10 mM further enhances both A. salmonicida growth in response to mucins and binding of the bacterium to mucins. In conclusion, GlcNAc and sialic acids are important determinants of the A. salmonicida interaction with its host at the mucosal surface. Furthermore, since the mucin glycan repertoire affects pathogen growth, the glycan repertoire may be a factor to take into account during breeding and selection of strains for aquaculture.

Atlantic Salmon Carries a Range of Novel O-Glycan Structures Differentially Localized on Skin and Intestinal Mucins.

Aquaculture is a growing industry, increasing the need for understanding host-pathogen interactions in fish. The skin and mucosal surfaces, covered by a mucus layer composed of mucins, is the first point of contact between fish and pathogens. Highly O-glycosylated mucins have been shown to be an important part of the defense against pathogens, and pathogens bind to host surfaces using lectin-like adhesins. However, knowledge of piscine O-glycosylation is very limited. We characterized mucin O-glycosylation of five freshwater acclimated Atlantic salmon, using mass spectrometry. Of the 109 O-glycans found, most were sialylated and differed in distribution among skin, pyloric ceca, and proximal and distal intestine. Skin O-glycans were shorter (2-6 residues) and less diverse (33 structures) than intestinal O-glycans (2-13 residues, 93 structures). Skin mucins carried O-glycan cores 1, 2, 3, and 5 and three types of sialic acids (Neu5Ac, Neu5Gc, and Kdn) and had sialyl-Tn as the predominant structure. Intestinal mucins carried only cores 1, 2, and 5, Neu5Ac was the only sialic acid present, and sialylated core 5 was the most dominant structure. This structural characterization can be used for identifying structures of putative importance in host-pathogen interactions for further testing in biological assays and disease intervention therapies.

Aeromonas salmonicida binds differentially to mucins isolated from skin and intestinal regions of Atlantic salmon in an N-acetylneuraminic acid-dependent manner.

Aeromonas salmonicida subsp. salmonicida infection, also known as furunculosis disease, is associated with high morbidity and mortality in salmonid aquaculture. The first line of defense the pathogen encounters is the mucus layer, which is predominantly comprised of secreted mucins. Here we isolated and characterized mucins from the skin and intestinal tract of healthy Atlantic salmon and studied how A. salmonicida bound to them. The mucins from the skin, pyloric ceca, and proximal and distal intestine mainly consisted of mucins soluble in chaotropic agents. The mucin density and mucin glycan chain length from the skin were lower than were seen with mucin from the intestinal tract. A. salmonicida bound to the mucins isolated from the intestinal tract to a greater extent than to the skin mucins. The mucins from the intestinal regions had higher levels of sialylation than the skin mucins. Desialylating intestinal mucins decreased A. salmonicida binding, whereas desialylation of skin mucins resulted in complete loss of binding. In line with this, A. salmonicida also bound better to mammalian mucins with high levels of sialylation, and N-acetylneuraminic acid appeared to be the sialic acid whose presence was imperative for binding. Thus, sialylated structures are important for A. salmonicida binding, suggesting a pivotal role for sialylation in mucosal defense. The marked differences in sialylation as well as A. salmonicida binding between the skin and intestinal tract suggest interorgan differences in the host-pathogen interaction and in the mucin defense against A. salmonicida.

Leptin triggers Ca(2+) imbalance in monocytes of overweight subjects.

Obesity is a major risk factor in numerous diseases, in which elevated intracellular Ca(2+) plays a major role in increased adiposity. We examined the difference between Ca(2+) signals in monocytes of lean and overweight subjects and the relationship between leptin induced NADPH oxidase activation and intracellular calcium concentration [Ca(2+)](i) homeostasis. Our results are as follows: (1) The basal level of [Ca(2+)](i) in resting monocytes of overweight subjects (OW monocytes) was higher than that in control cells, whereas the leptin-induced peak of the Ca(2+) signal was lower and the return to basal level was delayed. (2) Ca(2+) signals were more pronounced in OW monocytes than in control cells. (3) Using different inhibitors of cellular signaling, we found that in control cells the Ca(2+) signals originated from intracellular pools, whereas in OW cells they were generated predominantly by Ca(2+)-influx from medium. Finally, we found correlation between leptin induced superoxide anion generation and Ca(2+) signals. The disturbed [Ca(2+)](i) homeostasis in OW monocytes was fully restored in the presence of fluvastatin. Statins have pleiotropic effects involving the inhibition of free radical generation that may account for its beneficial effect on elevated [Ca(2+)](i) and consequently on the pathomechanism of obesity.

Decreased paraoxonase 1 (PON1) lactonase activity in hemodialyzed and renal transplanted patients. A novel cardiovascular biomarker in end-stage renal disease.

BACKGROUND: Human paraoxonase-1 (PON1) has also been described as a lactonase. Decreased PON1 lactonase activity was found to be a predictor of cardiovascular disease. Homocysteine thiolactonase activity may prevent proteins from homocysteinylation and is thought to be a protective factor against the progression of atherosclerosis. Previous studies have demonstrated decreased PON1 paraoxonase activity in hemodialyzed (HD) and renal transplant (TRX) patients; however, lactonase activity has not been investigated. We aimed to determine the paraoxonase and lactonase activities and to clarify the relationship between lactonase activity and a set of cardiovascular risk factors, such as homocysteine, cystatin C and asymmetric dimethylarginine (ADMA) levels, in HD and TRX patients and in healthy controls. METHODS: One hundred and eight HD and 78 TRX patients and 63 healthy controls were involved in the study. Paraoxonase and lactonase activities (paraoxon and gamma-thiobutyrolactone as substrates) were measured spectrophotometrically. ADMA level was determined with sandwich enzyme-linked immunosorbent assay. RESULTS: Both HD and TRX patients had significantly lower lactonase activities compared to the control group (P<0.05). Significantly lower paraoxonase activities were found in HD patients compared to the TRX group (P<0.05). Significant negative correlation was found between lactonase activity and ADMA level in the whole study population (P<0.001), while paraoxonase and lactonase activities showed significant positive correlation (P<0.001). Multiple regression analysis identified paraoxonase activity and homocysteine level as independent predictors of lactonase activity. CONCLUSION: Lactonase activity is a potential new predictor of cardiovascular risk in renal failure. Measurement of lactonase activity is recommended in future studies on HD and TRX patients.